Acute Oral Toxicity - Fixed Dose Procedure As per OECD Guideline 420

1. ACUTE TOXICITY STUDIES AS
PER OECD GUIDELINES
PREPARED BY :- KHUSHBOO THAKUR
M.PHARM SEM II
DEPARTMENT OF PHARAMACOLOGY
SSR COLLEGE OF PHARMACY, SAYLI, SILVASSA.

2. WHAT IS TOXICOLOGY ?
 Toxicology is the scientific study of adverse
effects that occur in living organisms due
to chemicals.
 It involves observing and reporting
symptoms, mechanisms, detection and
treatments of toxic substances, in
particular relation to the poisoning of
humans.

3. ADVERSE DRUG EFFECTS
 Any undesirable and/or unintended effects of drug
1. Predictable (type A reactions)
2. Non - predictable (type B reactions)
SIDE EFFECTS
Unwanted but often
unavoidable effects at
therapeutic doses.
SECONDARY
EFFECTS
Indirect consequences
of primary action of
drug.
TOXIC EFFECTS
Are result of excessive
pharmacological
effect of drug due to
over dosage or
prolonged use

4. ANIMAL TOXICITY TESTS
 Acute toxicity 14 days
 Sub-acute (repeated doses) toxicity 28 days
 Sub-chronic toxicity 3 months
 Chronic toxicity 6 months to 2 yrs.
 Special toxicity e.g.
Carcinogenicity

5. ACUTE ORAL TOXICITY
TEST GUIDELINE
401:
ACUTE ORAL
TOXICITY –
CONVENTIONAL
ACUTE
TOXICITY
420:
ACUTE ORAL
TOXICITY-
FIXED DOSE
PROCEDURE
423:
ACUTE ORAL
TOXICITY –
ACUTE TOXIC
CLASS METHOD
425:
ACUTE ORAL
TOXICITY – UP-
AND-DOWN
PROCEDURE

6. OECD GUIDELINES FOR ACUTE ORAL
TOXICITY
NUMBER TITLE ORIGINAL ADOPTION MOST RECENTLY
UPDATED
401 Acute oral toxicity-
Conventional acute
toxicity test
12 May, 1981 Date of deletion: 17
December 2002
420 Acute oral toxicity-
fixed dose procedure
17 July, 1992 8 February, 2002
423 Acute oral toxicity-
acute toxic class
method
22 March, 1996 8 February, 2002
425 Acute oral toxicity-
up and down
procedure
21 September, 1998 16 October, 2008

7. 420: Acute Oral Toxicity- Fixed Dose
Procedure
INTRODUCTION
 The original Guideline 420 was adopted in July 1992.
 It is the first alternative to the conventional acute toxicity test, described in
Test Guideline 401.
 Revision was considered timely –
 differ in harmonised LD50 Cut-off values
 Testing in one sex (usually females) is now considered sufficient

8.  Traditional methods for assessing acute toxicity use death of animals as an
endpoint.
 In 1984, a new approach to acute toxicity testing was suggested by the British
Toxicology Society -
 Approach avoided using death of animals as an endpoint, and relied instead on the observation of clear
signs of toxicity at one of a series of fixed dose levels.
 The statistical properties of the Fixed Dose Procedure have been evaluated using mathematical models
in a series of studies.

9.  Found way which- in vivo and modelling studies have demonstrated
 Uses fewer animals
 Causes less suffering
 Is reproducible
 Able to rank substance in a similar manner

10. INITIAL CONSIDERATIONS
 Use of only moderately toxic doses.
 Doses that are known to cause marked pain and distress, due to corrosive or
severely irritant actions, need not be administered.
 Moribund animals, or animals obviously in pain or showing signs of severe and
enduring distress shall be humanely killed (considered in the interpretation).

11.  The testing laboratory should consider all available information on the test
substance prior to conducting the study.
 Such information will include the following:
 identity and Chemical structure of test substance,
 Its physical and chemical properties,
 Results of any other in vivo or in vitro toxicity test on the substance,
 Toxicological data on structurally related substances or similar mixtures,
 Anticipated use of the substance.
 Helps to rank and classify acc. to “Globally Harmonised System” (GHS).

12. PRINCIPLE OF TEST
 Groups of animals of a single sex are dosed in stepwise procedure using the
fixed dose of 5, 50, 300 and 2000 mg/kg (exceptionally an additional fixed dose
of 5000 mg/kg may be considered).
 The initial dose level is selected on the basis of a sighting study.
 Clinical signs and conditions associated with pain, suffering, and impending
death.

13.  Groups of animals may be dosed at higher or lower fixed doses,
 It depend on the presence or absence of signs of the toxicity or mortality.
 Procedure continues until the
 Dose causing evident toxicity or no more than one death is identified,
 Or when no effects are seen at the highest dose
 Or when death occur at lowest dose.

14. DESCRIPTION OF THE METHOD
Selection of animal species
 Mostly rat is preferred as rodent species.
 Normally females are used.
 For males – adequate justification should be provided.
 Healthy young adult animals of commonly used laboratory strains should be
employed.
 Female should be nulliparous and non–pregnant.
 At the commencement of its dosing, should be between 8 to 12 weeks old, and
weight should fall in an interval within ±20% of the mean weight.

15.  Housing and feeding conditions
 Temperature = 22°C ± 3°C
 Relative Humidity = 50-60 %
 Light = Artificial (cycle of 12 hrs. light, 12 hrs. dark)
 Feeding = conventional lab. diets with unlimited drinking water supply
 Animal grouped = Group caging but no. of animals per cage must not interfere
with clear observations of each animal.

16.  Preparation of animals
 Randomly selected
 Marked to permit individual identification, and
 Kept in their cages for at least 5 days prior to start of dosing allow for
acclimatization.

17.  Preparation of doses
 Test substances should be administered in a constant volume over the range of
doses.
 In case of a liquid end product or mixture is to be tested – use undiluted test
substance i.e., at a constant concentration
 In either case, the maximum dose volume for administration must not exceeded
 In rodents maximum volume
 1 ml/100 gm of body wt.
 For Aqueous solution – 2 ml/100gm of body wt.

18.  Order of administration –
 Liquid
 Suspension
 Emulsion
 Vehicle other than water – toxicological characteristics should be known
 Preparation prior to administration

19. PROCEDURE
Administration of doses
 Test substance administered by gavage using a stomach tube or a suitable intubation
canula.
 Fasted overnight prior to dosing
 Weigh animal and administer the test substance
 Withheld food for further 3-4 hrs. in rats or 1-2 hrs. in mice

20.  Sighting study
 Purpose of the sighting study is to allow selection of the appropriate starting
dose for the main study.
 Test substance is administered to single animals in a sequential manner
following the flow charts.
 The starting dose for the sighting study is selected from the fixed dose levels of
5, 50, 300, and 2000 mg/kg as a dose expected to produce evident toxicity.

21.  Also, evidence from in vivo and in vitro data from the same chemical and from
structurally related chemicals.
 In absence of such information, the starting dose will be 300 mg/kg.
 At least 24 hrs. will be allow b/w the dosing of each animal.
 All animals should be observed for at least 14 days.

22.  In the case, where an animal tested at the lowest fixed dose level (5 mg/kg) in
the sighting study dies,
 The normal procedure is to terminate the study and assign the substance to GHS category I.
 For further confirmation use supplementary procedure.

25. MAIN STUDY
Numbers of animals and dose levels
 Select dose from sighting study.
 Perform study on a total of five animals of one sex at each level including
animals tested in sighting study.
 Time interval b/w dosing at each level determined by– Onset, duration and severity of
toxic signs
 Treatment of animals at the next dose should be delayed until one is confident
of survival of the previously dosed animals.

26.  To observe delayed toxicity – A period of 3 or 4 days b/w dosing at each dose
level is recommended.
 In case of inconclusive response – time interval may be adjusted as appropriate.
 Fixed dose of 5000 mg/kg – procedure outlined in Annex 4.

29.  Limit Test
 Performed when information indicating that the test material is likely to be
nontoxic. i.e., having toxicity only above regulatory limit doses
 Information about the toxicity of the test material gained from knowledge
about similar tested compound.
 Using the normal procedure test will be done.

30. OBSERVATIONS
 At least once during the first 30 min.
 Periodically during the first 24 hrs. with special attention given during first 4
hrs.
 And daily thereafter for a total of 14 days.
 However, duration of the observation should not be fixed rigidly.
 Times at which signs of toxicity appear or disappear are important, especially if
there is a tendency for toxic signs to be delayed.

31.  Observation should include
 Change in skin and fur, eyes, and mucous membranes, and
 Respiratory, circulatory, autonomic and central nervous systems, and somatomotor
activity and behaviour pattern.
 Attention should be directed to observation:
 Tremors,
 convulsions,
 salivation,
 diarrhoea,
 lethargy,
 sleep, and coma.

32.  Body weight
 Shortly before administration
 Thereafter weekly
 At the end

33.  Pathology
 All animals subjected to gross necropsy.
 Including those that die during the test or are removed from study

34. DATA AND REPORTING
Data
 Should include individual animal.
 All data summarised in tabular form, showing
 For each test group the number of animals used.
 The no. of animals displaying signs of toxicity.
 The no. of animals found dead during the test or killed for humane reasons.
 Time of death of individual animals.
 A description and the time course of toxic effects and reversibility, and
 Necropsy finding.

35.  Test report
 Test substance :
 Physical nature, purity, and where relevant, physic-chemical properties (including
isomerisation),
 identification data, including CAS No.
 Vehicle (if appropriate):
 Justification for choice of vehicle, if other than water

36.  Test animals:
 Species/strain used,
 Microbiological status of the animals, when known,
 No., age, and sex of animals (including, where appropriate, a rationale for males instead
of females),
 Source, housing conditions, diet, etc.
 Test report

37.  Test conditions:
 Details of test substance formulation, including details of the physical form of the
material administered,
 Detailed of the administration of the test substance including dosing volumes and time of
dosing,
 Details of food and water quality (including diet type/source, water source),
 The rationale for the selection of the starting dose.
 Test report

38.  Test report
 Results:
 Tabulation of response data and dose level for each animal (i.e., animals showing signs of
toxicity including mortality, nature, severity and duration of effects),
 Tabulation of body weight and body weight change,
 Individual weights of animals at the day of dosing, in weekly intervals thereafter, and at
time of death or sacrifice,
 Date and time of death if prior to scheduled sacrifice,
 Time course of onset of signs of toxicity and whether these were reversible for each
animal,
 Necropsy finding and histopathological findings for each animal, if available

39.  Test report
 Discussion and interpretation of results
 conclusions

40. EXCEPTIONAL TESTING (Dose >2000
mg/kg)
 If reliable evidence is already available that indicates the LD50 to be in the
range of category 5 values.
 Other animal studies or toxic effects in humans indicate a concern for human
health of an acute nature.
 Based on mortality incidences while testing schemes of Annex 3.
 Assignment to a more hazardous category is not warranted.

41.  Reliable information is available indicating significant toxic effects in humans,
 Any mortality is observed when tested up to category 4 values by oral route
 Where expert judgement confirms significant clinical signs of toxicity, when
tested up to category 4 values, except for diarrhoea, piloerection or an
ungroomed appearance,
 Where expert judgement confirms reliable information indicating the potential
for significant acute effects from the other animal studies.

42. Testing at dose above 2000 mg/kg
Sighting study
 Annex 2 are extended to include a 5000 mg/kg dose level
5000 mg/kg
A B C
Second animal to
be tested at 2000
mg/kg
Selection of 5000
mg/kg as the main
study starting dose
Selection of 5000
mg/kg as the main
study starting dose

43.  Main study
 Sequential procedure presented in annex 3 are extended to include a
5000mg/kg dose level
Main study
with 5000
mg/kg
A
(≥ 2 deaths)
B
(evident toxicity
and/or ≤ 1 death )
C
(no toxicity)
Testing of a 2nd
group at 2000
mg/kg
Substance being
unclassified acc. to
GHS
Substance being
unclassified acc. to
GHS